Under anaerobic growth conditions, Escherichia coli produces hydrogenase and this enzyme activity is involved in hydrogen uptake and hydrogen evolution. Previous studies from our laboratory have demonstrated that E. coli has at least two genes for hydrogenase activity (hydA and B), one gene for hydrogen uptake activity (hup) and a gene coding for an electron transport protein (fhl) coupling formate dehydrogenase-2 and hydrogenase in the formate hydrogenlyase reaction (Appendix). More recent studies revealed the presence of two additional hyd genes and one more fhl gene. Studies on the regulation of the hydrogen metabolism indicate that the synthesis of formate hydrogenlyase is regulated by a hitherto unindentified gene, fhlR and the hydrogen uptake is regulated by fnr gene product. We have purified a hydrogenase from E. coli and biochemicaly characterized this non-heme iron protein. The experiments proposed in this proposal are an extension of these studies and focussed toward understanding the molecular biology of the hydrogen metabolism. These experiments once completed will provide the following information: 1) The number of hydrogenase isoenzymes in the cell. Determine if there are two hydrogenase proteins catalyzing the two activities - hydrogen evolution and hydrogen uptake, or a single protein is catalyzing the two activities in association with different electron transport proteins. 2) The physical and biochemical properties of the hydrogenase(s). 3) The number and genetic location of hydrogenase gene(s) in the cell. 4) Minimum number of genes coding for accessory proteins required for activation of the apoprotein. 5) Regulation of hydrogenase synthesis - hydrogenase specific control systems and generalized control systems. Successful completion of these studies will help undelrstand the anaerobic metabolism in a cell and especially the way in which an anaerobic cell handles reducing power.